Evolutionary analysis of the functional plasticity of Staphylococcus aureus C₃₀ carotenoid synthase

Most natural carotenoids have 40-carbon (C₄₀) backbones, while some bacteria produce carotenoids with C₃₀ backbones. Carotenoid backbone synthases, the enzyme that catalyze the first committed step in carotenoid biosynthesis, are known to be highly specific. Previously, using C₃₀ backbone synthase (diapophytoene synthase, CrtM) from Staphylococcus aureus, we reported two size-shifting mutations, F26A and W38A, which confer C₄₀ synthase activity at the cost of the original C₃₀ synthase activity. In this study, we performed a directed evolution of the C₄₀-specialist variant CrtM_F26A in search of mutations that restore the original C₃₀ synthase function. Examination of the resultant mutants, together with the site-directed mutagenesis study identified three new mutations (H12A, D27A and I240F) that affect the size specificity of this enzyme. After re-defining the reading frame, we obtained CrtM variants that are highly active in C₃₀ and C₄₀ carotenoid synthesis.